ABSTRACT: Ovarian cancer, a significant contributor to cancer-related mortality, often exhibits limited responsiveness to hormonal therapies targeting the estrogen receptor (ESR1). This study aimed to elucidate the mechanisms behind ESR1 resistance to the therapeutic drug Fulvestrant (ICI182780 or ICI). We identified a 1.3MDa Megacomplex comprising ESR1, FOXA1, and PITX1 using size exclusion chromatography. These factors showed colocalized binding at 6,775 genomic positions in the ovarian cancer cell line, PEO4, including important super-enhancers. Notably, compared to the cytoplasmic version, nuclear ESR1 was minimally degraded by ICI, suggesting a mechanism for drug resistance via the protective confines of the Megacomplex. Combining a small molecule inhibitor of active chromatin and super-enhancers, JQ1, with ICI significantly enhanced ESR1 degradation, especially within the Megacomplex. Molecular analysis showed that this combination disrupted the cyto-nuclear ESR1 shuttling responsible for tumor cell proliferation. Pathway enrichment analysis of RNAseq gene sets following Estradiol, ICI, or ICI plus JQ1 treatments paralleled pathway enrichment found from target genes of Megacomplex binding identified through ChIPseq. Mass-spec analysis revealed that proteins within the Megacomplex macromolecule also produced similar pathway enrichments after modulation by Estradiol or ICI. Each of these findings independently implicates Megacomplex in ESR1-driven ovarian cancer chromatin regulation. Moreover, this combined treatment strategy exhibited superior inhibition of cell proliferation and viability compared to ICI alone. Other cell lines, PEO1, OVCAR4, OVCAR8, and the normal ovarian cell line, T1074, substantiated the specificity of the response. By uncovering ESR1's resistance within the Megacomplex, the combined ICI plus JQ1 treatment elucidates a novel drug treatment vulnerability.
